The present invention generally relates to a full length transcript promoter (FLt) sequence from mirabilis mosaic caulimovirus (MMV) and the use thereof to express chimeric genes in plant cells and plants. More particularly, the present invention relates to the use of wild type and modified MMV FLt DNA sequences, or multiple enhancer domains of the MMV FLt DNA sequences, to provide strong promoter activity to direct foreign gene expression in transgenic plants; a chimeric gene construct containing the MMV FLt DNA sequence (or multiple enhancer domains of the MMV FLt DNA sequence) and a coding sequence for a protein of interest; an expression vector containing the chimeric gene construct; a plant cell transformed with the chimeric gene construct; a plant seed transformed with the chimeric gene construct; a plant tissue transformed with the chimeric gene construct; and a transgenic plant transformed with the chimeric gene construct.
The Caulimoviruses and their Promoters for Plant Genetic Engineering
For plant genetic engineering applications, a number of strong, constitutive promoters have been derived from the genome of two pararetrovirus subgroups namely caulimovirus (Odell et al., 1985; Hasegawa et al., 1989; Sanger et al., 1990; Verdaguer et al., 1996; Maiti et al., 1997; Maiti and Shepherd, 1998) and badnavirus (Medberry et al., 1992; Bhattacharyya-Pakrassi et al., 1993). The caulimoviruses are a group of small circular DNA virus of approximately 8 kb pairs containing 6 to 8 open reading frames (ORFs). The cauliflower mosaic virus (CaMV) is one of the best characterized members of this group. Generally, two major transcript promoters are present in the caulimovirus genome: one is the pregenomic full-length transcript (similar to CaMV 35S transcript), which encompasses the whole genome and the other is the subgenomic transcript (similar to CaMV 19S transcript), which spans only gene VI region of caulimovirus. (Odell et al., 1981; Hasegawa et al., 1989; Driesen et al., 1993). In infected plants, the full-length transcripts (FLts) function both as mRNAs for synthesis of viral proteins and as templates during reverse-transcriptase mediated replication of viral genome (Guilley et al., 1982; Hull and Covey, 1983; Pfeiffer and Hohn, 1983). Transcriptional activity of the CaMV 35S promoter is the result of the combinatorial and synergistic interaction of different cis-elements present in the promoter sequence and the transacting nuclear binding protein factors (Benfey and Chua, 1990; Benfey et al., 1990a; 1990b; Fang et al., 1989).
The CaMV 35S promoter is a well-characterized strong constitutive promoter (Fang et al., 1989; Odell et al., 1985; Ow et al., 1987; Benfey et al., 1989, 1990; 1990a; 1990b; Lam, 1994) that has been extensively used for expressing foreign genes in monocotyledonous and dicotyledonous plants (Holtrof et al., 1995; Mitsuhara et al., 1996; Wilmink et al., 1995). The 35S promoter from CaMV is also active in microbes (Assaad and Singer, 1990; Pobjecky et al., 1990) and in animal cells (Zahm et al., 1989; Ballas et al., 1989).
It has been found that single or multiple copies of enhancer sequences from the CaMV 35S promoter can increase homologous and heterologous promoter expression in an orientation-independent manner (Ow et al., 1987; Kay et al., 1987; Omirulleh et al., 1993). Similar observation was made when single or multiple copies of the enhancer sequence are inserted upstream of the TATA-box of the CaMV 19S promoter (Ow et al., 1987; Driesen et al., 1993), rbcS-3A promoter (Fang et al., 1989) and the nos promoter (Odell et al., 1985). The duplication of enhancer sequences of FMV FLt-promoter (Maiti et al., 1997) and PC1SV-FLt-promoter (Maiti and Shepherd, 1998) also increase promoter activity.
Badnavirus, a subgroup of pararetrovirus, infects only monocotyledonous plants whereas caulimovirus infects dicotyledonous plants. Promoters from badnavirus like Commelana yellow mottle virus (CYMV) and rice tungro bacilliform virus (RTBV) are reported to be primarily active in vascular tissue (Bhattacharyya-Pakrasi et al., 1993; Medberry et al., 1992; Yin and Beachy, 1995).
The genomes of several caulimoviruses including CaMV (Gardner et al., 1981), carnation etched ring virus (CERV), (Hull et al., 1986), figwort mosaic virus (FMV), (Richins, 1987), soybean chlorotic mottle virus (SoCMV), (Hasegawa et al., 1989), peanut chlorotic streak virus (PC1SV), (Richins, 1993), cassava vein mosaic virus (CVMV), (Calvert et al., 1995), strawberry vein banding virus (SVBV), (Petrzik, 1996), and petunia vein clearing virus (PVCV), (Richert-Poggler and Shepherd, 1997) have been fully sequenced.
A number of transcriptional promoters have been derived from the genomes of pararetroviruses: for example the rice tungro bacilliform virus (RTBV), (Bhattacharyya-Pakrasi et al., 1993), the commelina yellow mottle virus (CYMV), (Medberry et al., 1992); the cauliflower mosaic virus (CaMV), (Lawton et al., 1987; Odell et al., 1985); the soya bean chlorotic mottle virus (SoyCMV), (Hasegawa et al., 1989); the figwort mosaic virus (FMV, strain DxS) (Gowda et al., 1989, Malti et al., 1997); FMV strain M3 (Sanger et al., 1990); the cassava vein mosaic virus (CVMV), (Verdaguer et al., 1996), and the peanut chlorotic streak virus (PC1SV), (Maiti and Shepherd, 1998).
Mirabilis mosaic virus (MMV), a member of the genus caulimovirus, has a circular double-stranded DNA genome of about 8 kb pairs with four single-stranded discontinuities in the DNA, one in the xcex1-strand and three in the complementary strand (Richins and Shepherd, 1983). It infects Mirabilis plant species (family Nyctaginaceae) generally found in warm parts of North America. The MMV was characterized as a member of caulimovirus based upon morphology of its virions and inclusion bodies (Brunt and Kitajima, 1973). MMV is serologically distinct from cauliflower mosaic virus, the type member of the genus (Brunt and Kitajima, 1973). The restriction enzyme map of the MMV genome is also quite different than that of other members of this genus (Richins and Shepherd, 1983).
The present invention is an MMV FLt promoter DNA sequence that can be used to direct and express selected foreign genes in plant cells and plants to confer useful properties therein. The present invention thus provides the isolation and characterization of the full length transcript (FLt) promoter from mirabilis mosaic caulimovirus.
The present invention is also optimal portions of the MMV FLt promoter DNA sequence that permit maximum promoter activity. In accordance with this embodiment, the present invention provides a 360 bp fragment (sequence xe2x88x92297 to +63 from the transcription start site (SEQ ID NO:4)) of the MMV FLt promoter DNA sequence.
The present invention is also the use of the MMV FLt promoter DNA sequence or optimal portions thereof in the form of a single sequence or in the form of multiple enhancer domains to express chimeric genes in plant cells and plants. The present invention is thus a chimeric gene construct containing the MMV FLt DNA sequence or a functional equivalent thereof (i.e., fragments thereof capable of promoter activity) in the form of a single MMV FLt DNA sequence or multiple enhancer domains of the MMV FLt DNA sequence and a coding sequence for a protein of interest; an expression vector containing the chimeric gene construct; a plant cell transformed with the chimeric gene construct; a plant seed transformed with the chimeric gene construct; a plant tissue transformed with the chimeric gene construct; and a transgenic plant transformed with the chimeric gene construct.
The present invention is also the use of the MMV FLt promoter DNA sequence, a functional equivalent thereof, in the form of a single sequence or multiple enhancer domains thereof, in combination with other promoters, e.g., FMV, PC1SV and CaMV 35S, having nonhomologous sequences to introduce two or more proteins of interest into plants and plant cells. The use of promoters having nonhomologous sequences avoids genetic instability due to recombination between identical promoter sequences.
The present invention is also the transcription start site (TSS) of the MMV FLt promoter as determined by primer extension analysis using total RNA isolated from transgenic plants containing a MMV promoter:uidA fusion gene. Analysis of the 5xe2x80x2 and 3xe2x80x2 deletion constructs show that an upstream region (sequence xe2x88x92248 to xe2x88x92193 from the transcription start site (SEQ ID NO:2)) is very important for the MMV FLt promoter activity along with as-1, TATA box regions. In addition, the present invention is a 31 bp sequence (+33 to +63 from the transcription start site (SEQ ID NO:3)) located downstream of a TATA box that provides for the maximum expression of the MMV FLt promoter.
Additional advantages of the present invention will be set forth in the description and examples that follow, or may be learned from practicing the invention. These and other advantages may be realized and attained by means of the features, instrumentalities and/or combinations particularly described herein. It is also to be understood that the foregoing general description and the following detailed description are only exemplary and explanatory and are not to be viewed as limiting or restricting the invention as claimed.
The invention itself, together with further advantages, will best be understood by reference to the following detailed description, taken in conjunction with the accompanying drawings.